Fig. 1: Characterization and validation of R26SAM/+ in mESCs.
a Schematic of R26LSL-SAM/+ and R26SAM/+. dCas9 is fused to VP64 with a nuclear localization sequence (NLS). MCP, P65, HSF1 are fused together with an intervening NLS sequence. The hybrid protein sequences are linked via a P2A peptide. Upon treatment with Cre recombinase, the loxP-stop-loxP cassette (LSL) is excised. b A representative western blot showing dCas9SAM (219 kDA) expression in targeted mESCs. dCas9SAM signals are normalized to actin (41 kDa). Three independent repeats have been completed with similar results. c Generalized schematic of R26 targeted guide arrays where “#a” indicates the number of activating guides included in the array and [Gene] refers to the target gene. Each guide is driven by a U6 promoter and separated by an extended RNA Pol III termination sequence. d Illustration of general SAM guide design approach. Guides are selected within 300 bases of the transcriptional start site (TSS) of each gene. e–h RNA-seq characterization of mESC lines harboring targeted gRNA arrays versus the parental R26SAM/+ line. The transcriptome of each clone was sequenced with five technical replicates to provide the number of target-specific transcripts per million (TPM) sequence reads. The target gene is noted with a blue star and the adjacent genes with red squares. Statistics: Pearson’s correlation coefficient was utilized to determine the r-value (e) r = 0.994, (f) r = 0.996, (g) r = 0.992, (h) r = 0.996.
